RNA-RNA interactions and the dynamic RNA conformations are important regulators in virus replication in several RNA virus systems and may also involved in the regulation of many important virus life cycle phases, including translation, replication, assembly, and switches in these important stages. The 5` non-translated region of Potato virus X (PVX) contains multiple cis-acting elements that facilitate various viral processes. It has previously been proposed that RNA-RNA interactions between various RNA elements present in PVX RNA genome are required for PVX RNA accumulation (Hu et al., 2007; Kim and Hemenway, 1999). This model was based on the potential base-pairing between conserved sequence elements at the upstream of subgenomic RNAs (sgRNAs) and at the 5` and 3` end of RNA genome. We now provide more evidence that RNA-RNA basepairing between elements present at the 5` end and upstream of each sgRNA is required for efficient replication of genomic and subgenomic plus-strand RNA accumulation. Site-directed mutations introduced at the 5` end of plus-strand RNA replication defective mutant (Δ12) increasing base-pairing possibility with conserved sequence elements located upstream of each sgRNAs restored genomic and subgenomic plus-strand RNA accumulation and caused symptom development in inoculated Nicotiana benthamiana plants. Serial passage of a deletion mutant (Δ8) caused more severe symptoms and restored wild type sequences and thus retained possible RNA-RNA base-pairing. Altogether, these results indicate that the RNA element located at the 5` end of PVX genome involved in RNA-RNA interactions and play a key role in high-level accumulation of plus-strand RNA in vivo.